Methods and apparatus for the location and concentration of polar analytes using an alternating electric field

ABSTRACT

A method is disclosed for effecting the concentration of a polar analyte in an alternating electric field. In the method, a relative translation of the polar analyte and an alternating electric field along a translation path is effected. A portion of the polar analyte is then trapped and concentrated in a concentration zone formed by the intersection of the translation path and the alternating electric field. Also disclosed are various devices for carrying out the forgoing method.

FIELD OF THE INVENTION

[0001] This invention relates to methods and apparatus for the locationand concentration of polar analytes using an alternating electric field.

BACKGROUND

[0002] A recent trend in the field of analytical instrumentation hasbeen the development of integrated microfluidic devices in whichmultiple operations are performed on a single device, e.g., Harrison etal., “Micromachining a Miniaturized Capillary Electrophoresis-BasedChemical Analysis System on a Chip,” Science, 261: 895 (1992). Suchdevices offer many advantages over conventional analytical formatsincluding the ability to handle very small volumes; ease and economy ofdevice fabrication; the ability to integrate multiple operations onto asingle integrated device; and the opportunity to achieve a high degreeof automation.

[0003] In many chemical and biochemical analysis methods performed usingmicrofluidic devices, it is advantageous to concentrate an analyte aspart of the analysis. For example, increased analyte concentrationgenerally leads to increased chemical reaction rates, increased rates ofmass transfer, and enhanced detectability. However, because conventionalconcentration methods require a solid phase pullout step (e.g.,adsorption), or a phase change of the analyte (e.g., precipitation), ora phase change of the solvent (e.g., evaporation), these methods are notwell adapted for use in a microfluidic device.

[0004] In addition, methods for controlling the location of an analyteare important in the design of methods using microfluidic devices. Forexample, prior to a separation step, it may be desirable to locate asample volume in a spatially-defined injection zone.

[0005] Therefore, it would be desirable to have a method for thelocation and concentration of an analyte that is well suited for use inintegrated microfluidic systems.

SUMMARY

[0006] The present invention is directed towards our discovery ofmethods and devices for the location and concentration of polar analytesusing an alternating electric field.

[0007] In a first aspect, the present invention provides a method forthe concentration of a polar analyte comprising the steps of effectingthe relative translation of the polar analyte and an alternatingelectric field along a translation path such that a portion of the polaranalyte is trapped and concentrated in a concentration zone formed bythe intersection of the translation path and the alternating electricfield.

[0008] In a second aspect, the present invention provides a device forthe concentration of a polar analyte comprising a translation path, afirst set of electrodes located to provide a first electric fieldeffective to cause the electrokinetic translation of a polar analytealong the translation path, and a second set of electrodes located toprovide an alternating second electric field intersecting thetranslation path and sufficient to trap and concentrate a portion of thepolar analyte in a concentration zone formed by the intersection of thetranslation path and the alternating second electric field.

[0009] It is a first object of the invention to provide a method for theconcentration of a polar analyte that does not require a solid-phasepull-out step or a phase change of the polar analyte or a solvent.

[0010] It is a second object of the invention to provide a method forthe manipulation or location of a polar analyte that does not require asolid-phase pull-out step or a phase change of the polar analyte or asolvent.

[0011] It is a third object of the invention to provide a method for theconcentration or manipulation of a polar analyte that does not requiremanual intervention and is therefore well suited to automation.

[0012] It is a fourth object of the invention to provide a method forthe concentration or manipulation of a polar analyte that is well suitedfor use in a microfluidic device.

[0013] The present invention will become better understood withreference to the following written description, drawings, and appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a schematic representation of a preferred methodaccording to the present invention.

[0015]FIG. 2A is a schematic representation of a preferred microfluidicconcentration and detection device according to the present invention.

[0016]FIG. 2B is a schematic representation of a preferred microfluidicconcentration, hybridization, and detection device according to thepresent invention.

[0017]FIG. 2C is a schematic representation of a preferred microfluidicconcentration device including a frit located in the concentration zone.

[0018]FIG. 3A is a schematic representation of a preferred microfluidicconcentration and electrokinetic separation device according to thepresent invention.

[0019]FIG. 3B is a schematic representation of an alternative preferredmicrofluidic concentration and electrokinetic separation deviceaccording to the present invention.

[0020]FIG. 4 is a schematic representation of several exemplaryelectrode configurations adapted for use in the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Reference will now be made in detail to certain preferredembodiments of the present invention, examples of which are illustratedin the accompanying drawings. While the invention will be described inconjunction with selected preferred embodiments, it will be understoodthat these embodiments are not intended to in any way limit the scope ofthe invention. On the contrary, the invention is intended to coveralternatives, modifications, and equivalents, which may be includedwithin the scope of the invention as determined by the appended claims.In addition, as used in this disclosure, the plural and singular numberswill each be deemed to include the other; “or” is not exclusive; and“includes” and “including” are not limiting.

[0022] Generally, the present invention comprises methods and apparatusfor locating and concentrating a polar analyte at a specified locationby effecting the relative translation of the polar analyte with respectto an alternating electric field along a translation path such that thepolar analyte is concentrated in a concentration zone formed by theintersection of the translation path and the alternating electric field.

[0023] The invention is based in part on the discovery that when a polaranalyte passes into an alternating electric field having the appropriatecharacteristics, the polar analyte will become trapped in aconcentration zone formed by the alternating electric field. While thetheoretical explanation for this highly unexpected and beneficialphenomena is not well understood, and the present invention is notintended to be limited in any way by the following or any othertheoretical explanation, it is thought by the inventors that thistrapping phenomenon is related to the Winslow effect, also referred toas the electrorheological effect, in which polar entities alignthemselves head-to-tail along the field lines of an electric fieldthereby forming filamentous structures, e.g., Winslow, J. AppliedPhysics (1947); and Winslow, U.S. Pat. No. 2,417,850.

[0024] I. Definitions

[0025] Unless stated otherwise, the following terms and phrases as usedherein are intended to have the following meanings:

[0026] “Translation path” means a path described as a result of arelative translation of an alternating electric field and a mediumpotentially containing a polar analyte. The translation path may haveany shape, e.g., straight, curved, and the like.

[0027] “Separation channel” means a channel used to conduct a separationprocess, e.g., separations based on electrokinetic, chromatographic, orother like process.

[0028] “Electrokinetic translation” means the movement of an entity inresponse to an electric field. Exemplary electrokinetic translationprocesses include but are not limited to electrophoresis,dielectrophoresis, electroendoosmosis, micellar electrokineticchromatography, isotachophoresis, and combinations of the foregoingprocesses.

[0029] “Electrokinetic translation system” means an apparatus effectiveto cause the electrokinetic translation of a polar analyte. Typically,an electrokinetic translation system will include a channel forsupporting a medium, a power supply, a set of two or more electrodes inelectrical communication with the channel, and electrical connectionsbetween the power supply and the two or more electrodes.

[0030] “Dipole moment” means the product q*R where q and −q are chargesof opposite polarity that are separated by a distance R. As used in thefollowing discussion, a dipole moment includes a permanent dipolemoment, a dipole moment resulting from the electric-field-inducedpolarization of an entity having a finite polarizability, a dipolemoment resulting from the orientation of an entity, e.g., orientationpolarization, a dipole moment resulting from the electric-field-inducedspace-charge distortion of a counter-ion atmosphere surrounding anentity, e.g., a Debye layer, or a dipole moment resulting from anycombination of the foregoing mechanisms.

[0031] “Alternating electric field” means an electric fieldcharacterized by a vector whose magnitude or direction varies with time.Included within the definition of an alternating electric field is amulti-phase electric field. The limits of the spatial extent of analternating electric field is that region in which the electric fieldstrength of the alternating electric field is greater than that requiredto trap and concentrate a polar analyte of interest with respect to thealternating electric field.

[0032] “Polar analyte” means an analyte having a dipole moment.

[0033] II. Methods

[0034] Referring to FIG. 1, in a preferred embodiment, the method of theinvention comprises a method for the location and concentration of apolar analyte 5 comprising the steps of effecting the relativetranslation of the polar analyte 5 along a translation path 10, andeffecting an alternating electric field 20 which intersects thetranslation path 10 such that some or all of the polar analyte 5 istrapped and concentrated in a concentration zone 30 formed by theintersection of the translation path 10 and the alternating electricfield 20.

[0035] The polar analyte 5 for use in the methods of the presentinvention may be any analyte having a dipole moment. The polar analytemay be charged or uncharged, and if the polar analyte is charged, it mayhave an overall net charge or be neutral. Preferably, the polar analytewill be present in a buffered electrolyte solution, more preferably anelectrolyte solution having a low ionic strength. Exemplary polaranalytes include nucleic acids, both single and double stranded,proteins, carbohydrates, viruses, cells, organelles, organic polymers,particles, and the like. A particularly preferred polar analyte for usein the methods of the present invention is single or double strandednucleic acid dissolved in an electrolyte.

[0036] The means used to effect the relative translation of the polaranalyte 5 with respect to the alternating electric field 20 along thetranslation path 10 may be any of a number of different means, orcombinations of means, capable of causing the polar analyte to becomelocated in a concentration zone 30. Such relative translation may beeffected by movement of the polar analyte 5, movement of the alternatingelectric field 20, or by movement of both the polar analyte 5 and thealternating electric field 20.

[0037] For example, the means used to effect the relative translation ofthe polar analyte with respect to the alternating electric field may besimple gravity forces. Alternatively, translation of the polar analyte 5may be induced by spinning the system 12 about a selected axis so as toimpose a centrifugal force having a component directed along thetranslation path 10. Instead, the polar analyte 5 may be caused to movealong the translation path 10 by capillary action or othersurface-mediate processes. Alternatively, active hydraulic pumping maybe employed to move the polar analyte 5 along the translation path as aresult of a pressure gradient, e.g., using a conventional microvolumesyringe pump. In yet another preferred embodiment where the polaranalyte is magnetic, a magnetic field may be used to cause thetranslation of the polar analyte along the translation path, e.g., usinga permanent magnet or an electromagnet. In an additional preferredembodiment of the subject invention, an electric field may be used toeffect the translation of the polar analyte by an electrokinetictranslation processes.

[0038] Where the means for effecting the relative translation of thepolar analyte with respect to the alternating electric field is effectedby movement of the alternating electric field 20, such movement may beachieved by mounting the electrodes used to form the alternatingelectric field on a moveable stage connected to a conventionalmechanical translation system. The mechanical translation system mayeffect the linear or non-linear translation of the electrodes. Suchmechanical translation systems are well known in the art, e.g.,Hunkapiller et al., U.S. Pat. No. 4,811,218; and Hueton et al., U.S.Pat. No. 5,459,325. Exemplary mechanical translation systems includeelectromechanical systems, e.g., a lead screw or belt drive connected toa motor, piezoelectric actuators, or pneumatic or hydraulic systems,e.g., a piston-in-cylinder drive system. One particularly preferredmechanical translation system comprises acomputer-controlled-DC-servo-motor-driven XY translation stage.

[0039] Regardless of the means used to effect the relative translationof the polar analyte with respect to the alternating electric fieldalong the translation path, during the concentration process, the forcesused to effect the relative translation must not be so strong so as tooverwhelm the trapping forces of the alternating electric field. Rather,the forces used to cause the relative translation along the translationpath should be balanced against the trapping forces used to trap thepolar analyte in the concentration zone as discussed in more detailbelow.

[0040] The alternating electric field 20 used to trap the polar analytecan be any alternating electric field effective to trap and concentratea polar analyte. Generally, the alternating electric field of theinvention may be characterized by a time vs. field strength profile, afrequency, and a maximum field strength. The properties of thealternating electric field required to trap the polar analyte willdepend on a number of easily accessible experimental parametersincluding the magnitude of the dipole moment of the polar analyte, thedielectric constant of the supporting medium, and, in the case of apolar analyte having an induced dipole moment, the polarizability of thepolar analyte or surrounding counterion atmosphere.

[0041] The time vs. field strength profile of the alternating electricfield may be sinusoidal, sawtooth, rectangular, superpositions of theforegoing, periodic or non-periodic, or any other profile capable ofbeing generated using a modem function generator, e.g., a Model 33120A15 MHz Function/Arbitrary Waveform Generator from Agilent Technologies.Preferably, the time vs. field strength profile of the alternatingelectric field is rectangular. The rectangular profile is preferredbecause it has essentially no zero-field component, resulting in a higheffective duty cycle. In a particularly preferred embodiment, the timevs. field strength profile of the alternating electric field is suchthat the time-averaged integrated field strength is zero where theaverage is taken over one complete cycle. This profile is preferredbecause it minimizes the extent to which a polar analyte located in theconcentration zone will be translated within the concentration zone in adirection other than along the translation path, and it reduces theamount of electrochemical reaction products produced at the surface ofthe electrodes, e.g., gas bubbles.

[0042] The frequency of the alternating electric field may be anyfrequency capable of trapping a portion of a polar analyte. However, formany polar analytes of practical importance, the frequency of thealternating electric field is preferably between about 10 Hz and 100megahertz (MHz), and more preferably between about 1 kilohertz (KHz) and100 KHz.

[0043] While the maximum field strength of the alternating electricfield may be any field strength suitable to a particular application,preferably, the maximum field strength of the alternating electricfield, as measured by the peak field strength of the alternatingelectric field, is between about 100 V/cm and 10,000 V/cm, and morepreferably between about 1,000 V/cm and 20,000 V/cm.

[0044] The alternating electric field may be spatially uniform orspatially non-uniform. For example, the alternating electric field maybe used to effect dielectrophoretic transport.

[0045] A trapped polar analyte may be released from a concentration zoneby either reducing the trapping strength of the alternating electricfield or by increasing the force used to effect the relative translationof the polar analyte with respect to the alternating electric field. Thetrapping strength of the alternating electric field may be modulated bychanging the frequency, field strength, or both. Alternatively, thepolar analyte can be released from the concentration zone by increasingthe forces used to effect the relative translation of the polar analyteand the alternating electric field, e.g., by increasing the electricfield used to drive electrokinetic translation of the polar analyte,increasing the pressure used to drive a pressure-driven flow of thepolar analyte, or increasing the rate of translation of the alternatingelectric field.

[0046] In a preferred embodiment, subsequent to an analyte concentrationstep according to the methods of the present invention, a furtheranalytical step is performed. In one preferred embodiment, after a polaranalyte has been concentrated in a concentration zone, the polar analyteis directed into an analytical separation process, for example anelectrokinetic or chromatographic separation process. The concentrationand localization methods of the present invention are particularlyadvantageous where the subsequent analytical separation process iselectrophoresis because the pre-separation concentration step canprovide a concentrated and narrow injection zone leading to bothincreased separation performance, e.g., decreased plate height, andenhanced detectability of the separated components.

[0047] In a preferred embodiment of the present invention in which thepolar analyte is a nucleic acid, subsequent to or during theconcentration of the analyte nucleic acid in the concentration zone, thenucleic acid analyte is subjected to a nucleic acid hybridizationreaction in which the concentrated nucleic acid analyte is contactedwith one or more complementary nucleic acids under conditions suitablefor sequence-specific hybridization. In a particularly preferredembodiment, the complementary nucleic acids are bound to a solidsupport, e.g., an array of support-bound nucleic acids including one ormore potentially complementary nucleic acids. The support-bound nucleicacids may be synthetic polynucleotide probes, cDNA molecules, or anyother nucleic acid or nucleic acid analog capable of sequence-specifichybridization. Exemplary arrays of support-bound nucleic acids aredescribed elsewhere, e.g., Singh-Gasson et al., Nature Biotechnology,17: 974-978 (1999); Blanchard and Friend, Nature Biotechnology, 17: 953(1999); Brown et al., U.S. Pat. No. 5,807,522. The pre-hybridizationconcentration step of the present invention may result in an increasedrate of hybridization, the ability to use a less concentrated sample, oran enhancement of the detectability of the products of the hybridizationreaction. While this embodiment has been described in the context ofnucleic acid hybridization, it will be apparent to one skilled in theart of biochemical instrumentation and analysis that this embodimentcould be equally applied to other process in which a polar analyte iscontacted with a binding complement, e.g., antibody-antigen pairs,receptor-ligand pairs, biotin-avidin pairs, and the like.

[0048] In yet another preferred embodiment of the methods of the presentinvention, during or subsequent to the concentration of the polaranalyte in a concentration zone, the polar analyte is detected using adetector, for example a fluorescence detector. In this embodiment, thepre-detection concentration step can lead to enhanced detectability ofthe polar analyte thereby leading to a more sensitive measurement, orthe opportunity to use a less sophisticated or expensive detectionsystem, e.g., UV absorbence rather than laser-induced fluorescence.Where detection takes place during the concentration process, theconcentration process may be monitored in real time.

[0049] In yet another preferred embodiment of the invention, during orsubsequent to the concentration of the polar analyte, the concentratedpolar analyte is contacted with a reactant and a chemical reaction iseffected between the reactant and the concentrated polar analyte. Suchreactions may include chemical, immunological, or enzymatic processes,e.g., analyte labeling, protein digestion, DNA digestion orfragmentation, DNA synthesis, and the like. The concentration step maylead to an increased reaction rate or enhanced detectability of thereaction products.

[0050] III. Devices

[0051] As is evident based on the foregoing discussion of the variouspreferred methods according to the present invention, a wide variety ofdevices may be constructed to carry out the methods. The particularelements, lay-out, dimensions, materials, and experimental conditionsused to construct and operate the devices of the invention may varydepending on the particular method or application being addressed.

[0052] Generally, a device according to the present invention includes ameans for generating an alternating electric field and a means foreffecting the relative translation of a polar analyte with respect tothe alternating electric field along a translation path. The device isconstructed so that in operation a concentration zone is formed at theintersection of the translation path and the alternating electric field.Several exemplary devices according to the present invention aredescribed below.

[0053]FIG. 2A shows a schematic representation of an exemplarymicrofluidic concentration and detection device 119 according to thepresent invention. The device 119 comprises an analyte loading reservoir120, a translation channel 130, and a waste reservoir 125, such that theanalyte loading reservoir 120 is in fluid communication with the wastereservoir 125 through the translation channel 130. Both the analyteloading reservoir 120 and the waste reservoir 125 contain electrodes 150a and 150 b for effecting the electrokinetic translation of an analytealong the translation channel 130. The electrodes 150 a and 150 b areeach connected to a power supply 170 for providing a voltage differencebetween electrodes 150 a and 150 b and thereby effecting an electricfield along the translation channel 130 sufficient to cause theelectrokinetic translation of an analyte along the translation channel130. The device 119 further comprises a second pair of electrodes 140 aand 140 b located at opposite sides of the translation channel 130 so asto effect an alternating electric field across the width of thetranslation channel 130. The electrodes 140 a and 140 b are connected toan alternating field power supply 160 for providing a time-variantvoltage difference between electrodes 140 a and 140 b and therebyeffecting an alternating electric field substantially across thetranslation channel 130. A concentration zone 181 is located betweenelectrodes 140 a and 140 b. Optionally, the device 119 further includesa detector (not shown) positioned such that material located in theconcentration zone 181 may be detected. In addition, the device 119further optionally includes a computer 180 connected to the power supply170, the alternating field power supply 160, and to the detector (notshown) to control and monitor the operation of the device and to managethe acquisition, analysis, and presentation of data.

[0054]FIG. 2B shows a variant of the device described in FIG. 2A 191 inwhich a polynucleotide hybridization array 192, or an array of any othersupport-bound binding moiety, is located in the concentration zone 181located between electrodes 140 a and 140 b. The location of thehybridization array 192 is such that an analyte to be contacted with thearray is concentrated in the concentration zone 181 adjacent to thearray prior to hybridization in order to increase the rate of masstransfer of the analyte to the array and enhance the detectability ofthe hybridized analyte.

[0055] In operation, the device 191 works generally as follows. Thetranslation channel 130 and the waste reservoir 125 are filled with asupporting medium, e.g., a low ionic strength buffered electrolytesolution, and a polar analyte is placed in the analyte loading reservoir120, e.g., using a conventional micropipette. Then, power supply 170 isactivated thereby causing the electrokinetic translation of the analyteout of the analyte loading reservoir 120 and into the translationchannel 130. Next, before the analyte reaches the concentration zone 181located generally between pair of electrodes 140 a and 140 b, thealternating field power supply 160 is activated. As the analyte movesthrough the translation channel 130 into the concentration zone 181formed by the intersection of the alternating electric field and thetranslation channel 130, the polar analyte is trapped and concentratedin the concentration zone. This process is continued until the desiredamount of analyte is located in the concentration zone. Finally, theamount of polar analyte in the concentration zone is detected by thedetector. Alternatively, the detector monitors the concentration zonethroughout the process in order to detect the time course of theconcentration process. Or, in the device 191 shown in FIG. 2B, theconcentration process is continued until the concentration of the polaranalyte is sufficient to effect the hybridization step.

[0056]FIG. 2C shows another variant of the device described in FIG. 2A192 in which a frit 193 is located in the concentration zone 181 locatedbetween electrodes 140 a and 140 b. While the theoretical explanationfor this highly unexpected and beneficial phenomena is not wellunderstood, and the present invention is not intended to be limited inany way by the following or any other theoretical explanation, it isthought by the inventors that the frit 193 creates spatialnonuniformities in the alternating electric field which, in certaincircumstances, serves to enhance the concentration effect of theinvention. The frit 193 has a porous structure comprising through-porescontaining a fluid medium such that material can be transported throughthe frit. Preferably, the pore structure of the frit comprises poreshaving effective internal diameters of between 0.5 and 50 μm.

[0057] In one embodiment, the frit is made up of an insulating matrixsuch that the AC electrical conductivity of the insulating matrix issubstantially less than the AC electrical conductivity of the fluidmedium contained in the pores of the frit. Preferably, the electricalconductivity of the fluid medium is 3 times greater than that of theinsulating matrix, and more preferably between 10 and 1000 times greaterthan that of the insulating matrix. The insulating matrix may be formedfrom any insulating material that can be fabricated into a frit.Exemplary materials include plastics, ceramics, and the like.Preferably, the insulating matrix is a plastic such aspolymethylmethacrylate.

[0058] In an alternative embodiment, the frit includeselectrically-conductive particles suspended in the insulating matrixsuch that there exists a plurality of electrically isolated regions eachcontaining one or more conductive particles. In an ideal case, eachparticle would be electrically isolated from all other particles by theinsulating matrix. The conductive particles may be formed from anyconductive or semi-conductive material, but preferably the particles aremetallic, e.g., silver, gold, platinum, copper, and the like, orsemiconductor materials, e.g., gallium arcinide. In a preferredembodiment, the particles are substantially spherical and have adiameter of between about 0.5 μm to about 50 μm.

[0059] Methods for fabricating frits according to the present inventionare well known. Briefly, one exemplary preferred frit fabricationprocedure is as follows. Dissolve 1 gram of Plexiglass in 250 ml ofchloroform at room temperature to form a Plexiglass/chloroform mixture.Next, mix 20 grams of 150 mesh (99.5%) copper basis copper particles(Alfa AESAR, stock # 10160) with 2 ml of the plexiglass/chloroformmixture to form a particle suspension. The metal particles should beapproximately 2-10 μm in diameter. Mixing should be performed at roomtemperature by swirling the suspension until it becomes evenly mixed forapproximately 5 minutes. To form the frit, aspirate a 2-5 mm length ofthe mixture into a micropipet (Micropipetts calibrated color codeddisposable, VWR Scientific, cat # 53432-921, Size 100 ul ) using aconventional pipette bulb. Allow the aspirated suspension to drypartially for approximately 5 minutes and then push the suspensionfarther into the micropipette using a rigid wire or rod. Finally, allowthe frit to dry for an additional 30 minutes at room temperature. Thefrit may then be used in situ or be mechanically removed from themicropipette.

[0060]FIG. 3A shows a schematic representation of another exemplarymicrofluidic device 200 according to the present invention. The device200 comprises an analyte loading reservoir 205, an electrokineticseparation channel 225, and a waste reservoir 210, such that the analyteloading reservoir 205 is in fluid communication with the waste reservoir210 through the electrokinetic separation channel 225. The analyteloading reservoir 205 and the waste reservoir 210 contain electrodes 215a and 215 b, respectively, for effecting an electrokinetic translationof a polar analyte along the electrokinetic separation channel 225.Electrodes 215 a and to 215 b are connected to power supply 240. Thedevice further comprises trapping electrodes 220 a and to 220 b foreffecting an alternating electric field between electrodes 220 a and 220b. Electrodes 220 a and to 220 b are connected to alternating-voltagepower supply 230. The device further comprises a detector 245 located ata distal end of the electrokinetic separation channel 225 relative tothe loading reservoir 205 for detecting the polar analyte in a detectionzone 246 after the electrokinetic separation is substantially complete.Optionally, the device 200 includes a computer 235 which is connected topower supply 230, power supply 240, and detector 245, to control andmonitor the operation of the device and to manage acquisition, analysis,and presentation of data. The device 200 may optionally further includea second alternating voltage power supply and pair of electrodes (notshown) located proximate to the detection zone 246 for trapping andconcentrating the separated components of the polar analyte prior to orduring detection in order to further increase the detectability of theseparated components.

[0061] In operation, the device 200 works as follows. First, theelectrokinetic separation channel 225 and the waste reservoir 210 arefilled with an electrolyte solution capable of supporting theelectrokinetic translation of the polar analyte along the electrokineticseparation channel 225. Then, the analyte loading reservoir 205 isfilled with an analyte, for example using a conventional micropipette.Next, power supply 240 is activated in a forward polarity to initiatethe electrokinetic translation of the polar analyte along theelectrokinetic separation channel 225. In addition, power supply 230 isactivated in order to establish the alternating electric field used totrap and concentrate the polar analyte in the concentration zone 181located approximately between electrodes 220 a and to 220 b. Optionally,to increase the amount of analyte trapped in the concentration zone, thepolarity of power supply 240 may be cycled between the forward polarityand a reverse polarity such that the polar analyte is translated backand forth through the concentration zone. Once a sufficient amount ofthe polar analyte is located and concentrated in the concentration zone,the power supply 240 is activated in the reverse polarity therebydrawing uncaptured material out of the electrokinetic separation channel225 and back in to loading reservoir 205. At this point, any analyteremaining in analyte loading reservoir 205 may be removed and replacedwith an appropriate electrolyte solution. Next, power supply 230 isturned off and the electrokinetic separation process is continued alongthe electrokinetic separation channel 225 until the components of thepolar analyte are transported to the detection zone 246 and detected bythe detector 245.

[0062]FIG. 3B shows a schematic representation of yet another exemplarymicrofluidic device 420 according to the present invention. The device420 is a variant of the device 200 of FIG. 3A in which the singleanalyte loading reservoir 205 in device 200 is replaced with a pair ofreservoirs: an analyte loading reservoir 405 and an analyte wastereservoir 410. The analyte loading reservoir 405 contains electrode 400and the analyte waste reservoir 410 contains electrode 415. Analyteloading reservoir 405 is connected to the electrokinetic separationchannel 225 by branch channel 406, and analyte waste reservoir 410 isconnected to electrokinetic separation channel 225 by branch channel407. Other elements of the device 420 are as described with respect todevice 200 in FIG. 3A.

[0063] In operation, the device 420 works as follows. First, theelectrokinetic separation channel 225, the waste reservoir 210, thebranch channels 407 and 406, and the analyte waste reservoir 410 arefilled with an electrolyte solution capable of supporting theelectrokinetic translation of the polar analyte along the electrokineticseparation channel 225. Then, the analyte loading reservoir 205 isfilled with an analyte, for example using a conventional micropipette.Next, power supply 240 is activated in a forward polarity to effect apotential difference between electrodes 400 and 215 b, to initiate theelectrokinetic translation of the polar analyte through branch channel406 and along the electrokinetic separation channel 225 and intoconcentration zone 181. As before, power supply 230 is activated inorder to establish the alternating electric field used to trap andconcentrate the polar analyte in the concentration zone 181 locatedsubstantially between electrodes 220 a and to 220 b. Optionally, thepolarity of power supply 240 may be cycled between the forward polarityand a reverse polarity such that the polar analyte is translated backand forth through the concentration zone. Once a sufficient amount ofthe polar analyte is located and concentrated in the concentration zone,the power supply 240 is activated in the reverse polarity to effect apotential difference between electrodes 415, 400 and 215 b such thatuncaptured material is drawn out of the electrokinetic separationchannel 225 and the analyte loading reservoir 405 and into the analytewaste reservoir 410. Optionally, after sweeping the uncaptured analyteinto analyte waste reservoir 410, analyte present in waste reservoir 415may be removed and replaced with an appropriate electrolyte solution.Next, power supply 230 is turned off and the electrokinetic separationprocess is continued along the electrokinetic separation channel 225between electrodes 400 and 215 b until the components of the polaranalyte are transported to the detection zone 246 and detected by thedetector 245.

[0064] The fluidic channels used in many of the preferred devices of theinvention may be any channel capable of supporting a sample containing apolar analyte and solvent or other supporting media required to carryout a method of the invention. The channels may be discrete, for exampleindividual capillary tubes, or formed as part of an integratedmicrofluidic device, e.g., channels etched in a glass substrate.Preferably, the fluidic channels are formed as part of an integratedmicrofluidic device including one or more intersecting channels andreservoirs. Exemplary microfluidic devices, and several alternativemethods for device fabrication, are disclosed in Soane and Soane, U.S.Pat. Nos. 5,750,015 and 5,126,022; Manz, U.S. Pat. Nos. 5,296,114 and5,180,480; Junkichi et al., U.S. Pat. No. 5,132,012; and Pace, U.S. Pat.No. 4,908,112. Other references describing microfluidic devices includeHarrison et al., Science, 261: 895 (1992); Jacobsen et al., Anal. Chem.66: 2949 (1994); Effenhauser et al., Anal. Chem. 66:2949 (1994); andWoolley and Mathies, P.N.A.S. USA, 91:11348 (1994). A general discussionof microfabrication techniques is provided by Madou in Fundamentals ofMicrofabrication, CRC Press, Boca Raton, Fla. (1997).

[0065] The fluidic channels may be present in the device in a variety ofconfigurations, depending on the particular application being addressed.The internal volume of a channel will preferably range from about 1 nlto about 10 μl, and more preferably from about 10 nl to about 2 μl. Thelength of a channel will generally range from about 1 mm to about 50 cm,usually between about 5 cm to 30 cm. However, in certain applications,channels may have a length up to or greater than 100 cm, e.g., where thechannel is used as an electrokinetic or chromatographic separationchannel. The cross-sectional dimensions (e.g., width, height, diameter)will range from about 1 μm to about 400 μm, usually from about 20 μm toabout 200 μm. The cross-sectional shape of the channel may be anycross-sectional shape, including but not limited to circular, ellipsoid,rectangular, trapezoidal, square, or combinations of the foregoingshapes. The fluidic channels may be straight, serpentine, helical,spiral, or any other configuration, depending on the requirements of aparticular application. For example, if it is desired to construct along fluidic channel in a small substrate, it may be advantageous toconstruct a channel having a spiral or serpentine shape.

[0066] The fluidic channels of the device may optionally comprise, andusually will comprise, fluid reservoirs at one or both termini, i.e.,either end, of the channels. Where reservoirs are provided, they mayserve a variety of purposes including a means for introducing variousfluids into the channel, e.g., buffer, elution solvent, sieving media,reagent, nnse or wash solutions; means for receiving waste fluid from achannel, e.g., an electrokinetic flowpath; or, as electrode reservoirsfor contacting an electrode with an electrolyte and for supplying ionsto support an electrokinetic process. Generally, the reservoirs willhave a volume of between 1 μl and 100 μl, preferably between about 1 μland 10 μl. Larger reservoirs may be desirable if the reservoirs servemultiple fluidic channels.

[0067] The subject devices may also optionally comprise an interfacesystem for assisting in the introduction of an analyte into the device.For example, where the analyte is to be introduced into the device usinga syringe, the interface system may comprise a syringe interface whichserves as a guide for the syringe needle into the device, e.g., as aseal over an analyte introduction reservoir.

[0068] Depending on the particular application, configuration, andmaterials from which the device is fabricated, a detection region fordetecting the presence of a particular analyte may be included in thedevice, e.g., element 246 in FIG. 3. For example, preferably at leastone region of the electrokinetic channel includes a detection regionthat is fabricated from a material that is optically transparent,generally allowing light of wavelengths ranging from 180 to 1500 nm,usually 250 to 800 nm, to be transmitted through the material with lowtransmission losses, i.e., less than about 20%, preferably less thanabout 5%. Suitable optically transparent materials include fused silica,certain optically-clear plastics, quartz glass, borosilicate glass, andthe like.

[0069] The subject devices according to the present invention may befabricated from a wide variety of materials, including glass, fusedsilica, acrylics, thermoplastics, silicon, and the like. Preferably, thematerials will have high dielectric breakdown potential, e.g., greaterthan about 100 kV/cm, be mechanically rigid, be chemically compatiblewith the polar analyte and any associated solvents or media, and have alow dielectric loss factor, e.g., less than about 0.05 at 1 MHz. Thevarious components of the integrated device may be fabricated from thesame or different materials, depending on the particular use of thedevice, the economic concerns, solvent compatibility, optical claritycolor, mechanical strength, mechanical features, electrical properties,thermal properties, and the like. For example, a planar substratecomprising microfluidic flowpaths and a cover plate may be fabricatedfrom the same material, e.g., polymethylmethacrylate (PMMA), ordifferent materials, e.g., a substrate of PMMA and a cover plate ofglass. For applications where it is desired to have a disposableintegrated device, due to ease of manufacture and cost of materials, thedevice will typically be fabricated from a plastic. For ease ofdetection and fabrication, the entire device may be fabricated from aplastic material that is optically transparent with respect to theoptical wavelengths used for detection. Also of interest in certainapplications are plastics having low surface charge under conditions ofelectrophoresis. Particular plastics useful for the fabrication ofdisposable devices according to the present invention include but arenot limited to polymethylmethacrylate, polycarbonate, polyethyleneterepthalate, polystyrene or styrene copolymers.

[0070] Optionally, the surface properties of materials used to fabricatethe devices may be altered in order to control analyte-wallinteractions, electroendoosmosis, bonding properties, or any othersurface-mediated property of the materials. The surface modificationsmay be based on covalent attachment of coating agents, or physicalattachment, e.g., by ionic, hydrophobic, or van der Waals interactions.Exemplary surface modification techniques are described elsewhere, e.g.,Hjerten, U.S. Pat. No. 4,680,201; Cobb et al., Anal. Chem., 62:2478-2483 (1990); van Alstine et al., U.S. Pat. No. 4,690,749; andBelder and Schomburg, Journal of High Resolution Chromatography, 15:686-693 (1992).

[0071] The devices according to the present invention may be fabricatedusing any convenient means used for fabricating like devices. In apreferred embodiment of the invention, conventional molding and castingtechniques are used to fabricate the devices. For example, for devicesprepared from a plastic material, a silicon mold master which is anegative for the channel structure in the planar substrate of the devicecan be prepared by etching or laser micromachining. In addition tohaving a raised ridge which will form the channel in the substrate, thesilica mold may have a raised area which will provide for a cavity intothe planar substrate for forming reservoirs or other fluidic features.Where convenient, the procedures described in U.S. Pat. No. 5,110,514may be employed. A cover plate may be sealed to the substrate using anyconvenient means, including ultrasonic welding, adhesives, etc.

[0072] Alternatively, the devices may be fabricated usingphotolithographic techniques as employed in the production ofmicroelectronic computer chips as follows. First, a substrate supportsuch as a polymethylmethacrylate card approximately the size of aconventional credit card is provided. The surface of the card itself isnot electrically conducting nor is the card. On the card is firstdeposited a thin layer of an electrically conducting material, e.g., ametal. The coating may be applied by a variety of different techniquesknown to those skilled in the art and may be comprised of a variety ofdifferent types of materials provided they are capable of conductingelectricity and preferably chemically inert, e.g., platinum, gold, andthe like. The layer is preferably thin; on the order of 100 angstroms toa few microns of thickness. The electrically conducting layer is thencoated with a layer of material which is both light-sensitive andnon-conducting. Once the light-sensitive, non-conducting layercompletely covers the electrically conducting layer, a mask is appliedto the surface of the light-sensitive, non-conducting layer. After themask covers the layer, it is exposed to light resulting in a pattern ofportions of the light-sensitive material being solvent soluble andportions being solvent insoluble. The soluble portions are washed awayand the exposed electrically conducting material etched away leavingtraces of wires and connectors to the wires under the insoluble portionof the light sensitive material. The underlying electrically conductingmaterial will provide electrode connections to the fluidic channels andreservoirs. By removing a portion of the insoluble material from theends or connectors of the electrically conducting traces remaining fromthe electrically conducting layer, electrical connection can be madewith the electrode connectors to the fluidic elements. In addition theelectrode traces are protected from wear and abrasion by the protectivecoating. As will be apparent to those skilled in the art, the maskutilized in the above production procedure can be produced so as toprovide essentially an unlimited number of different electrodeconnections to the device, e.g., see S. M. Sze, VLSI Technology, SecondEdition, McGraw-Hill, New York, N.Y. (1988).

[0073] Where the devices are formed from glass, standard etching processmay be used, e.g., K. Fluri et al., Anal. Chem., 66: 4285-4290 (1996);Z. Fan and D. J. Harrison, Anal. Chem., 66: 177-184 (1994).

[0074] Alternatively, rather than utilizing the photolithographic ormolding and casting techniques generally described above to fabricatethe devices of the present invention, it is possible to utilize otherfabrication techniques such as employing various types of lasertechnologies and/or other technologies such as silk-screening and vapordeposition which make it possible to provide extremely small (in size)and large numbers of electrodes and fluidic channels, or lamination, orextrusion techniques.

[0075] Generally, the fluidic channels will contain a supporting mediumto support the analyte. The medium may be an organic solvent, a buffersolution, a polymeric solution, a surfactant micellular dispersion, orgel of the type generally used in connection with biochemical separationtechniques. Preferably, the medium will have a low ionic strength, e.g.,below about 100 mM salt, more preferably below about 10 mM salt, andcomprise a solvent having a low dielectric constant, e.g., below about80. A particularly preferred medium comprises an entangled aqueouspolymer solution, e.g., Madabhushi et al, U.S. Pat. No. 5,567,292.

[0076] The electrodes used in the devices of the invention, e.g., toeffect the alternating electric field, may be fabricated usingconventional methods from conventional materials. Exemplary methods forelectrode fabrication include photolithography, silkscreen techniques,and a simple wire electrode. Preferably, the materials used to form theelectrodes are electrically conductive and chemically inert.Particularly preferred materials include gold, platinum, tungsten, andthe like.

[0077] Preferably the electrodes used to effect the alternating electricfield have a shape which serves to form an electric field that resultsin a single, well-defined concentration zone having the desireddimensions. FIG. 4 shows several exemplary electrode geometries; 300,305, 310, 315, and 320. The spacing of the electrodes is chosen so as,togenerate an electric field having sufficient strength to trap a polaranalyte of interest but not so high as to cause excessive bubbleformation at the electrodes, e.g., see Washizu et al., IEEE Transactionson Industry Applications, 30 (4): 835-843 (1994). Generally, the spacingbetween the electrodes is preferably between about 50 μm and 2 mm.

[0078] Preferably, the electrodes used to effect the alternatingelectric field do not protrude into the translation path or otherwisecome into direct physical contact with the translation path. Rather, theelectrodes are preferably positioned such that the alternating electricfield produced between the electrodes intersects the translation path,but the electrodes themselves are physically removed from thetranslation path, e.g., using an isolation layer or an electricalconnection through a conductive bridge. Exemplary materials useful forforming an isolation layer include SiO₂, Al₂O₃, polyimide, diamond,glass, and the like. Isolating the electrodes from the translation pathis preferred because it serves to minimize the extent to which theelectrodes may become fouled by the analytes or any other materialpresent in the translation path, or become pasiviated due to oxidationor other chemical transformation of the electrode surface.

[0079] The means for effecting an alternating electric field may includeany conventional alternating current power supply using conventionalelectrical connectors. An exemplary alternating current power supplysuitable for use in the present invention is a Model 33120A 15 MHzFunction/Arbitrary Waveform Generator function generator from AgilentTechnologies. The means for effecting an alternating electric fieldfurther includes the above-described electrodes and electricalconnections between the electrodes and the alternating current powersupply.

[0080] The electrodes may be fabricated and integrated into the devicesof the invention using any one of a number of conventional techniques.For example, wire electrodes may be mechanically attached to the device,electrodes may be incorporated into the device during a lamination stepor during a casting step, electrodes may be deposited onto the deviceusing plasma deposition or electrochemical deposition techniques.

[0081] As discussed above, certain embodiments of the present inventioninclude a detection system for detecting an analyte during or subsequentto an analysis. Any sort of conventional detection system may be usedwith the present invention, including systems for measuringfluorescence, radioactivity, optical absorbence, fluorescencepolarization, electrical conductivity, electrochemical properties,refractive index, and the like. A particularly preferred detectionsystem employs laser-excited fluorescence.

[0082] In a preferred embodiment of the present invention, the channels,reservoirs, or concentration zone of the microfluidic device aremaintained at a controlled temperature using a temperature controlsystem. For example, temperature control may be desirable in order toincrease the reproducibility of an analysis, control the properties of amedium or analyte, or to speed up an analysis. A preferred temperaturecontrol system may include a heating element, e.g., a resistive heatingelement in combination with a fan, a cooling device, e.g., a Peltierdevice or other conventional refrigeration device, an enclosedthermally-isolated chamber, one or more temperature measurement sensors,and a programmable feedback control system. Preferably, the temperaturecontrol system is connected to a computer for controlling and monitoringthe overall system.

[0083] The devices of the present invention may be used in combinationwith robotic systems to automate certain steps of a process, e.g., arobot may be used to manipulate a plurality of microfluidic devices forhigh-throughput multi-device applications, or to introduce an analyteinto a device of the invention, or to transfer a medium into or out ofthe device. Such robots may be any kind of conventional laboratoryrobot, preferably with fluid-handling capability, e.g., a Beckman BioMeksystem.

[0084] In certain embodiments of the present invention, a computer isused to monitor or control various aspects of the device including:controlling the operation of the components of the device, e.g., powersupplies, temperature controller, pumps, etc.; controlling systemsassociated with the device, e.g., laboratory robots; monitoring theperformance of the device, e.g., measuring electric field strengths,temperatures, or pressures; managing of data acquisition, data analysis,and data presentation activities; or providing a convenient userinterface for the programmable operation of the device. The computer ofthe present invention may be any type of conventional programmableelectronic computer, e.g., a personal computer. The computer may beconnected to other elements of a system through conventional devices,e.g., an A/D converter.

[0085] All publications, patents, and patent applications mentionedherein are hereby incorporated by reference to the same extent as ifeach individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

[0086] Although only a few embodiments have been described in detailabove, those having ordinary skill in the art of chemicalinstrumentation will clearly understand that many modifications arepossible in the preferred embodiments without departing from theteachings thereof. All such modifications are intended to be encompassedwithin the scope of the following claims.

We claim:
 1. A method for the location and concentration of a polaranalyte comprising the steps of: effecting the relative translation ofthe polar analyte and an alternating electric field along a translationpath such that a portion of the polar analyte is trapped andconcentrated in a concentration zone formed by the intersection of thetranslation path and the alternating electric field.
 2. The method ofclaim 1 wherein the polar analyte is charged.
 3. The method of claim 2wherein the polar analyte comprises a nucleic acid.
 4. The method ofclaim 1 wherein the relative translation is effected by movement of thealternating electric field.
 5. The method of claim 1 wherein therelative translation is effected by movement of the polar analyte. 6.The method of claim 5 wherein the relative translation is effected bythe electrokinetic translation of the polar analyte.
 7. The method ofclaim 1 wherein a time-vs.-field strength profile of the alternatingelectric field is rectangular.
 8. The method of claim 1 wherein thetime-averaged integrated field strength of the alternating electricfield taken over one complete cycle is zero.
 9. The method of claim 1wherein the frequency of the alternating electric field is between about10 Hz and 100 MHz.
 10. The method of claim 1 wherein the frequency ofalternating electric field is between about 1 KHz and 100 KHz.
 11. Themethod of claim 1 wherein the maximum field strength of the alternatingelectric field is between 100 V/cm and 100,000 V/cm.
 12. The method ofclaim 1 wherein the maximum field strength of the alternating electricis between 1,000 V/cm and 20,000 V/cm.
 13. The method of claim 1 whereinthe polar analyte is a nucleic acid and further comprising the step ofperforming a nucleic acid hybridization reaction between the polaranalyte and a complementary nucleic acid during or subsequent to theconcentration of the polar analyte.
 14. The method of claim 13 whereinthe complementary nucleic acid is bound to a solid support.
 15. Themethod of claim 1 further comprising the step of detecting the polaranalyte during or subsequent to concentration.
 16. The method of claim 1further comprising the step of performing a chemical reaction betweenthe polar analyte and a reactant in the concentration zone during orsubsequent to the concentration of the polar analyte.
 17. The method ofclaim 1 further comprising the step of releasing the polar analyte fromthe concentration zone.
 18. The method of claim 17 further comprisingthe step of performing an analytical separation process subsequent tothe step of releasing the polar analyte from the concentration zone. 19.The method of claim 18 wherein the analytical separation process is anelectrokinetic separation process.
 20. The method of claim 19 whereinthe electrokinetic separation process is electrophoresis.
 21. A methodfor the location and concentration of a polar analyte comprising thesteps of: locating the polar analyte in an elongate channel having atranslation path; effecting the translation of the polar analyte alongthe translation path; effecting an alternating electric field sufficientto trap and concentrate all or part of the polar analyte in aconcentration zone formed by the intersection of the translation pathand the alternating electric field.
 22. A device for the location andconcentration of a polar analyte comprising: a means for effecting analternating electric field; a means for effecting the relativetranslation of a polar analyte with respect to the alternating electricfield along a translation path; wherein the alternating electric fieldis sufficient to trap and concentrate a portion of the polar analyte ina concentration zone formed by the intersection of the translation pathand the alternating electric field.
 23. A device for the location andconcentration of a polar analyte comprising: a translation path; a firstset of electrodes located to provide a first electric field effective tocause the electrokinetic translation of a polar analyte along thetranslation path; and a second set of electrodes located to provide analternating second electric field intersecting the translation path andsufficient to trap and concentrate a portion of the polar analyte in aconcentration zone formed by the intersection of the translation pathand the alternating second electric field.
 24. A device for the locationand concentration of a polar analyte comprising: a translation path; oneor more electrodes located to provide an alternating electric fieldintersecting the translation path and sufficient to trap and concentratea portion of a polar analyte located in a concentration zone formed bythe intersection of the translation path and the alternating electricfield; and a means for effecting the relative translation of the polaranalyte with respect to the alternating electric field along thetranslation path.
 25. The device of claim 24 further including adetector positioned to detect material located in the concentrationzone.
 26. The device of claim 24 further including an array ofsupport-bound binding complements located in the concentration zone. 27.The device of claim 26 wherein the polar analyte is nucleic acid and thesupport-bound binding complement is a complementary nucleic acid. 28.The device of claim 24 further including a frit located in theconcentration zone wherein the frit is made of an insulating matrix. 29.The device of claim 28 wherein the AC electrical conductivity of theinsulating matrix is less than the AC electrical conductivity of a fluidmedium located in pores of the frit.
 30. The device of claim 29 whereinthe AC electrical conductivity of the insulating matrix is at least 3times less than that of the fluid medium.
 31. The device of claim 29wherein the AC electrical conductivity of the insulating matrix isbetween about 10 and 1000 times less than that of the fluid medium. 32.The device of claim 28 further including electrically-conductiveparticles suspended in the insulating matrix such that there exists aplurality of electrically isolated regions each containing one or moreconductive particles.
 33. The device of claim 24 further including aseparation channel in fluid communication with the concentration zone.34. The device of claim 24 further including a temperature controlsystem in thermal communication with the concentration zone.